Skip to main content
. 2010 Mar 11;29(8):1377–1388. doi: 10.1038/emboj.2010.30

Figure 4.

Figure 4

Formation of vessel structures in differentiating 2D EBs involves both VEGFR2 and VEGFR3. (A) Expression of VEGFRs in 2D EB vascular structures. EBs differentiating in 2D cultures for 12 days in the presence of VEGFA shows vessel-like structures co-expressing VEGFR2 and VEGFR3 (upper panels; merged immunostainings to the right), or VE-cadherin and VEGFR3 (lower panels; merged immunostainings to the right). Scale bar=50 μm (upper), 100 μm (lower). (B) Complex formation between VEGFRs in 2D EBs. Left: Immunoprecipitation (ip) of VEGFR3 from day 12 EBs treated with vehicle (–) VEGFA (A) or VEGFC (C) for 15 min followed by immunoblotting for VEGFR2 (upper left panel). Immunoblotting for VEGFR3 (lower left panel) shows equal loading of VEGFR3. Right: Parallel aliquots of cell lysate were analysed by immunoprecipitation of VEGFR2 followed by immunoblotting for VEGFR3 (upper right panel). VEGFR2 immunoblotting (lower right panel) showed equal loading. (C) Heterodimers in CD31-positive cells. Formation of VEGFR2/-3 heterodimers as detected by in situ PLA (red spots) on 2D EBs immunostained for CD31 (green), in response to vehicle (–), VEGFA or VEGFC. Scale bar=10 μm. (D) Quantification of PLA spots in CD31-positive cells as in C. n=8. (E) Identification of LYVE1-positive cells. EBs in 2D cultures were treated with VEGFA or VEGFC until day 12, and immunostained to detect expression of CD31 (green) and LYVE1 (red). Panels to the right show merged immunostainings. Lymphatic vascular structures expressing LYVE1 but not CD31 are indicated by arrows in the VEGFC-treated cultures. Single, rather than vessel-organized LYVE1-positive cells, are indicated by asterisk. Scale bar=100 μm. (F) Heterodimers in LYVE1-positive cells. EBs in 2D culture were treated as indicated above and processed for immunostaining to detect CD31 (blue) and LYVE1 (white), followed by in situ PLA to detect VEGFR2/-3 heterodimers (red). Scale bar=10 μm. (G) Quantification of PLA spots in LYVE1-positive cells as in (F). n=8. Asterisks in panels D and G indicate the degree of significance (**P<0.01, ***P<0.001).